The Problem: The dental pulp contains pluripotent stem cells that play a critical role in tooth development and tissue regeneration. We have recently demonstrated that human dental pulp stem cels diferentiate into vascular endothelial cells that organize themselves into functional blood vessels in vivo. This exciting observation suggest that dental pulp stem cells can give rise to blood vessels that support the high metabolic demands of "tissue-making", in addition to differentiate into the actual cells that generate the new tissue (e.g. odontoblasts, osteoblasts). However, the mechanisms that control the vasculogenic fate of dental pulp stem cells are not understood. Such knowledge is required to maximize the use of the diferentiation potential of dental pulp stem cells in clinical applications. Hypothesis: In preliminary studies, we demonstrated that vascular endothelial growth factor (VEGF) enhances the vasculogenic potential of dental pulp stem cells. However, the signaling events required for VEGF-induced differentiation of dental pulp stem cells into endothelial cells are unknown. It is known that the Wingless (Wnt) signaling pathway plays a critical role in the determination of cell fate during development. Notably, Wnt inhibits the differentiation of dental pulp stem cells into odontoblasts. But the role of Wnt signaling in directing dental pulp stem cells towards a vasculogenic fate is not understood. Interestingly, a tight correlation between vasculogenesis and bone formation is observed during development. However, the impact of stem cel- mediated vasculogenesis on dentin formation is not known. Here, we will use the tooth as an experimental model for the evaluation of mechanisms that regulate the commitment of stem cells towards the vasculogenic phenotype and determine the role of stem cell-derived blood vessels in mineralized tissue formation. The mechanistic hypothesis of this proposal is: "VEGF and Wnt signaling regulate the vasculogenic fate of dental pulp stem cells". To test this hypothesis, we propose the following specific aims: -Specific Aim #1: To study mechanisms involved in VEGF-induced differentiation of dental pulp stem cells into endothelial cells. -Specific Aim #2: To evaluate the function of Wnt signaling on the determination of dental pulp stem cell fate. -Specific Aim #3: To understand the functional relation between vascular differentiation of dental pulp stem cells and dentinogenesis. Significance: The clinical translation of stem cell-based therapies requires the understanding of mechanisms that control the differentiation fate of these cels. This project aims at the development of mechanism-based approaches that exploit the vasculogenic potential of stem cells to provide the blood vessels required for the generation of new tissues and organs. Our ultimate goal is to employ a deeper understanding of the biology of mesenchymal stem cells of dental origin to benefit patients that require tissue regeneration. PUBLIC HEALTH RELEVANCE: The dental pulp is a readily accessible source of stem cells that are suitable for tissue regeneration. One of the critical limitations to tissue engineering is the absolute requirement of appropriate vascularization to provide oxygen and nutrients for cells engaged in the making of new tissues. This project is centered on the study of mechanisms involved in the determination of the vasculogenic fate of dental pulp stem cells. The ability of controlling the processes that regulate the differentiation of stem cells into blood vessels constitutes a major step forward towards the use of stem cell-based therapies in patients that need tissue regeneration.